Ability of the MeltPro MTB/PZA Assay to Detect Susceptibility to Pyrazinamide in Rifampin-Resistant Tuberculosis Patients

ABSTRACT Prediction of susceptibility to pyrazinamide (PZA) directly from sputum has been challenging. The MeltPro MTB/PZA assay, based on melting curve analysis, can simultaneously detect Mycobacterium tuberculosis and the resistance to PZA from sputum. We aimed to evaluate the MeltPro MTB/PZA assay to predict PZA resistance among rifampin-resistant tuberculosis (RR-TB) patients. We prospectively enrolled RR-TB patients in the registered trials, and their baseline sputum samples were obtained to perform the assay and culture. DNA sequencing of culture isolates was analyzed and used as the reference standard. Sanger sequencing was performed for samples with discrepant results between next-generation sequencing (NGS) and the investigational assay. The main analysis was conducted in the population of patients with interpretable results by both NGS and the assay. A total of 239 patients with RR-TB were screened, and 220 underwent the MeltPro MTB/PZA assay. The assay provided no information for 25 of 220 patients (11.4%). Among the remaining 195 patients, 13 had negative culture or insufficient raw NGS sequencing data, and 15 had indeterminate assay results. A total of 167 patients were included in the main analysis. Against DNA sequencing, the sensitivity, specificity, and negative predictive value of the assay for detecting resistance to PZA were 91.4% (95% confidence interval [CI], 87.1% to 95.6%), 89.9% (95% CI, 85.3% to 94.5%), and 95.2% (95% CI, 91.9% to 98.4%), respectively. In conclusion, the MeltPro MTB/PZA assay is a fast semiautomatic molecular platform to rapidly predict resistance to PZA from sputum and holds promise as a screening tool with satisfactory sensitivity. IMPORTANCE This study evaluated the accuracy of the MeltPro MTB/PZA assay at detecting the presence of PZA resistance through registered clinical trials. Compared to DNA sequencing, the assay had high sensitivity and negative predictive value, suggesting its potential utility as a screening tool in clinical practice. The assay could serve as an ideal primary screening tool in low PZA-resistant M. tuberculosis prevalence settings and could be used as an additional test to identify PZA resistance rapidly and initially in the RR-TB population.


INTERPRETATION OF THE RESULTS Ⅰ. REFERENCE VALUE
The controls in the kit must meet the following requirements, otherwise, the experiment will be considered invalid. The Tm value range of positive control (wild type) in each channel of A, B, and C reaction is shown as follows:
Reaction D only gives amplification signals. For positive control and negative control, their Ct values range are as follows: Positive control: FAM channel: Ct value is less than 18.5 (Ct<18.5); HEX channel: Ct value is less than 27.0 (Ct<27.0). Negative control: FAM channel: No Ct; HEX channel: Ct value is less than 27.0 (Ct<27.0). Always calibrate the above Tm values with the positive control (wild-type) for each run.
The Tm values will be given automatically by the instrument.

Ⅱ. EXPLANATION OF RESULTS
1. Interpretation of positive control: For each run, the Tm value of positive control of all channels should be within the range of reference values, otherwise the experiment will be considered invalid. 2. Interpretation of negative control: There should be no signal observed for negative control in each channel except D-HEX, which indicates that there is no contamination during DNA extraction or detection. Otherwise the experiment will be considered invalid.
3. Interpretation of specimens: 1) Whether a specimen is a mutant or not is determined by comparing the difference in Tm values between the specimen and the positive control: -△Tm= Tm (positive control) -Tm(specimen) -Wild peak: -1.6°C＜△Tm＜1.6°C -Mutant peak: |△Tm|≥1.6°C The result is valid when the △Tm≥1.6°C of the third melting peak (△Tm3) in any channel, it is suggested to check the sample quality; or repeat test is recommended to confirm the result. 2) For each specimen, the interpretation of the results is as follows: A. When there are no missing melting peaks (the total number of melting peaks is more than or equal to 36): a. When there are three wild peaks in each of the 12 channels of reaction A, B, and C without any mutant peak, it is determined as wild type (sensitive to pyrazinamide, Figure S2). ② If three or more melting peaks are missing in the 12 channels of reaction A, B, or C, and the sequence number of missing melting peaks is continuous, there may be a short deletion in the target sequence, which means that the sample is resistant to pyrazinamide; if the missing melting peaks is discontinuous, the resistance of this sample to pyrazinamide is unconfirmed. b. When the Ct≥25.2 in the D-FAM channel and the Ct<27.0 in the D-HEX channel, it indicates that the sample concentration is too low to be detected, and the resistance to pyrazinamide is unconfirmed. A repeat sample extraction is recommended to confirm the result. c. When the Ct≥27.0 or no signal in the D-HEX channel, it is indicated that the sample may contain a PCR inhibitor. Purification or appropriate dilution (e.g., 5 times dilution) of the template is recommended in this situation. C. Invalid result: except for the above cases, a result may be invalid if no melting peak is shown in one or more channels. The possible reasons are: (a.) No or low MTB in the samples; (b.) Wrong operation; (c.) The kit is ineffective.
Warning: Environmental pollution in the laboratory, reagent contamination, and crosscontamination of specimens will lead to false-positive; improper transportation and/or storage, and incorrect reagent preparation may lower the accuracy of the test and lead to a false negative or inaccurate detection results.